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It is difficult to visualize histological details on semi-ultrathin sections by light microscopy after immunohistochemical labeling because the histological structures in such sections cannot be distinguished by standard counter-staining. To solve this problem and to visualize the immunoreactivity specific for various antigens, we have developed a technique that involves a combination of immunofluorescent staining of semi-ultrathin sections of epoxy resin-embedded samples and either the corresponding differential interference contrast (DIC) images or the corresponding images in transmission mode obtained by confocal laser-scanning microscopy (LSM), providing detailed information about the immuno-localization of histological and cellular structures. Haraguchi and Yokota (2002) were the first to describe a similar method, including treatment of sections with 10% sodium ethoxide to remove epoxy resin (Litwin et al., 1984) and reaction with fluorescence-labeled second antibodies. In our method, we use 10% sodium methoxide to remove epoxy resin from sections (Mayor et al., 1961). There is no significant difference between the two treatments, but Haraguchi & Yokota (2002) used fluorescence-labeled second antibodies while we use a combination of biotin-conjugated second antibodies and streptavidin fluorescence. Our preliminary experiments indicated that the latter system has greater sensitivity than the former in the case of semi-ultrathin sections. Using our above-mentioned method, we are now easily able to detect immunofluorescence on semi-ultrathin sections of epoxy resin-embedded specimens. To demonstrate the effectiveness of our method, in the present study we examined the immunofluorescence of immuno-stained keratins and collagens and the images obtained, in transmission mode, of the lingual mucosa of the filiform and circumvallate papillary areas of tongues of fetus and juvenile rats by LSM. By combining images, we were able to define clearly the histological locations of keratin 13 (K13) and keratin 14 (K14) in the lingual epithelium (Iwasaki et al., 2006a, 2011a) and those of collagen II (CII) and collagen III (CIII) in the lingual connective tissue (Asami et al., 2008, Iwasaki et al., 2008, 2011b). Furthermore, we demonstrated that our newly developed technique for localization of pairs of antigens should be useful for investigations of very small specimens, such as fetal tissues and organs (Aoyagi et al., 2008).

Fluorescence Immunohistochemistry by Confocal Laser-Scanning Microscopy for Studies of Semi-Ultrathin Specimens of Epoxy Resin-Embedded Samples